Immune effector cells pre-infected with oncolytic virus

ABSTRACT

Compositions and methods are provided for the treatment of cancer. An immune effector cell population is pre-infected with an oncolytic virus. The combined therapeutic is safe and highly effective, producing an enhanced anti-tumor effect compared to either therapy alone. The methods of the invention thus provide for a synergistic effect based on the combined biotherapeutics.

This invention was made with Government support under contract P01 CA49605-14 awarded by the National Cancer Institute. The Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Neoplasia is a process that occurs in cancer, by which the normalcontrolling mechanisms that regulate cell growth and differentiation areimpaired, resulting in progressive growth. This impairment of controlmechanisms allows a tumor to enlarge and occupy spaces in vital areas ofthe body. If the tumor invades surrounding tissue and is transported todistant sites it will likely result in death of the individual.

The desired goal of cancer therapy is to kill cancer cellspreferentially, without having a deleterious effect on normal cells.Several methods have been used in an attempt to reach this goal,including surgery, radiation therapy, and chemotherapy.

Local treatments, such as radiation therapy and surgery, offer a way ofreducing the tumor mass in regions of the body that are accessiblethrough surgical techniques or high doses of radiation therapy. However,more effective local therapies with fewer side effects are needed.Moreover, these treatments are not applicable to the destruction ofwidely disseminated or circulating tumor cells eventually found in mostcancer patients. To combat the spread of tumor cells, systemic therapiesare used.

The primary weapon against cancer is chemotherapy. However,chemotherapeutic agents are limited in their effectiveness for treatingmany cancer types, including many common solid tumors. This failure isin part due to the intrinsic or acquired drug resistance of many tumorcells. Another drawback to the use of chemotherapeutic agents is theirsevere side effects. These include bone marrow suppression, nausea,vomiting, hair loss, and ulcerations in the mouth.

Proposed alternative therapies include the administration of oncolyticviruses, and the use of viral vectors to deliver a transgene whoseexpression product activates a chemotherapeutic agent. The geneticengineering of viruses for use as oncolytic agents has initially focusedon the use of replication-incompetent viruses. This strategy was hopedto prevent damage to non-tumor cells by the viruses. A major limitationof this approach is that these replication-incompetent viruses require ahelper virus to be able to integrate and/or replicate in a host cell.These viruses are limited in their effectiveness, because eachreplication-defective retrovirus particle can enter only a single celland cannot productively infect others thereafter. Therefore, they cannotspread far from the producer cell, and are unable to completelypenetrate many tumors in vivo. More recently, genetic engineering ofoncolytic viruses has focused on the generation of“replication-conditional” viruses in an attempt to avoid systemicinfection, while allowing the virus to spread to other tumor cells.Replication-conditional viruses are designed to preferentially replicatein actively dividing cells, such as tumor cells. Thus, these virusesshould target tumor cells for oncolysis, and replicate in these cells sothat the virus can spread to other tumor cells.

However, while the virus-based approach has provided evidence ofsignificant therapeutic effects in animal models of tumors, the methodis limited by the efficiency of viral infection; the requirement of ahelper virus or producer cell line for some viral vectors; tumor cellheterogeneity for the cellular factor(s) complementing viral mutantgrowth for other viral vectors; and antiviral immune responses.

A variety of immune cell-based cancer therapies have also been proposed,many of which rely on the identification of tumor-associated antigensthat are often weak or expressed on only a subset of tumor cells.Cytokine induced killer (CIK) cells are a population of cells derivedfrom human PBMC's following ex vivo expansion with γIFN, anti-CD3antibody and IL-2. They bear phenotypic markers of NK and T cells,express NKG2D and have been found to mediate killing of tumor cellsthrough recognition of a class of stress-associated ligands expressed onthe tumor cell surface (NKG2D ligands). CIK cells therefore do not relyon specific antigens and they have also been shown to target a varietyof tumors and exert their cytotoxic effects following systemic delivery.Previous pre-clinical imaging studies found that at 72 hours (h) afterintravenous delivery signals from CIK cells were found primarily at thetumor site. However, tumor cell killing required effector to targetratios of five to ten CIK cells per tumor cell in vitro, and adependence on over expression of NKG2D ligands on the tumor targets.

Targeted biological therapies hold tremendous potential for thetreatment of cancers, yet their effective use has been limited byconstraints on delivery and effective tumor targeting. There exists aneed for a local therapy that provides for effective killing of tumorcells. The present invention addresses this need.

Relevant Literature

Leemhuis et al. (2005) Biol Blood Marrow Transplant 11, 181-7 (2005); Lu& Negrin (1994) J Immunol 153, 1687-96; Kim et al. (2001) Nat Med 7,781-7 (2001). Thorne & Kim (2004) Expert Opin Biol Ther 4, 1307-21.Puhlmann et al. (2000) Cancer Gene Ther 7, 66-73 (2000). Hamerman et al.(2005) Curr Opin Immunol 17, 29-35.

SUMMARY OF THE INVENTION

Methods are provided for the treatment of cancer, through administrationto a patient of an effector cell population that is pre-infected with anoncolytic virus. The effector cells are preferably T cells, which may beautologous or allogeneic. In some embodiments, the cells are cytokineinduced killer cells, which do not rely on recognition through the Tcell antigen receptor for cytoxicity. In other embodiments, the cellsare tumor infiltrating T lymphocytes. The effector cells are infectedwith an oncolytic virus, preferably a replication competent virus, e.g.vaccinia, adenovirus, etc. Oncolytic viruses of interest arereplication-selective or tropism modified viruses that are either onlycapable of entering into, or of completing a successful infection cyclewithin, transformed cells.

Pre-infection of effector cells with oncolytic virus resulted in aprolonged eclipse period where the virus remained within the cells untilinteraction with, and infiltration into, the tumor. The infected cellsare preferably administered to the patient during the eclipse phase. Inthe combined therapeutic, the effector cells were shown to retain theirability to traffic to tumors. At the tumor site the oncolytic virus wasreleased deep in the tumor rather than merely at the surface; thus thecell mediated delivery of the virus led to enhanced biodistributionwithin the tumor. In addition, the cytotoxic effects of the effectorcells may be increased by viral replication in the tumor target. Thiscombined therapeutic has been demonstrated to be safe, with minimalviral infection of normal tissues, and highly effective, producing anenhanced anti-tumor effect compared to either therapy alone. The methodsof the invention thus provide for a synergistic effect based on thecombined biotherapeutics.

Other objects and advantages of the present invention will becomeevident from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thedetailed description of the invention when considered in connection withthe accompanying drawings which show as follows:

FIG. 1A-1C. Vaccinia virus displays unusual replication kinetics in CIKcells. (a) Viral replication was followed for different vaccinia strainsin CIK cells after infection at a multiplicity of infection (MOI) of 1.0plaque forming units (PFU)/cell (*p=0.034, T12 test, SEM). (b) Viralreplication of the same vaccinia strains in the B-cell lymphoma cellline OCI-ly8 (MOI of 1.0 PFU/cell). (c) Kinetics of replication of vvDDin CIK cells following infection at an MOI of 1.0 PFU/cell. Viralinfectious units (PFU/ml) were titered separately in the cell and mediafractions of the infected plate at times indicated. Cell-associated andcell-free viral titers are plotted to reveal viral replication kinetics.CIK cell numbers within infected or uninfected plates were counted atthe same time points.

FIG. 2A-2C. CIK cells retain their ability to kill target cells despitepre-infection with vvDD, and infection of CIK-resistant target cellswith vvDD increases their susceptibility to CIK-mediated killing due toNKG2D ligand expression. (a) UCI-101 target cells expressing luciferasewere mixed with different numbers of CIK effector cells (ratios of100:1, 50:1, 20:1 and 10:1 or target only). CIK effector cells had beenpre-infected with vvDD for 24-h, 48-h or 72-h (MOI 1.0 PFU/cell).Luciferase output, as an indication of cytotoxicity, was measured usingan IVIS50 system (Xenogen Corp.) after 4 h. (b) SKOV-3 target cellsexpressing luciferase, alone or pre-infected with vvDD (MOI 1.0PFU/cell) for 24-h, were mixed with CIK cells at effector:target ratiosof 20:1. Luciferase signal was measured after 4 h (p=0.0076, T-test,SEM). (c) UCI-101 or SKOV-3 cells alone or pre-infected with vvDD (MOI1.0 PFU/ml for 8 h) were stained with anti-MICA/MICB antibody conjugatedto PE and the numbers of cells expressing these cell surface markerswere determined by FACS analysis. Isotype controls produced less than 1%positive staining.

FIG. 3A-3D. Biodistribution of vvDD expressing luciferase delivered tomice bearing subcutaneous UCI-101 tumors either alone or within CIKcells and immunofluorescence microscopy of tumor sections (UCI-101). (a)Mice received 1×10₇ CIK cells pre-infected with vvDD-luc (MOI 1.0PFU/cell) via tail vein injection (left) or 1×10₇ PFU vvDD-luc via tailvein injection (right). Injections occurred on day 0. Animals wereimaged at the times indicated (in days post therapy) using an IVIS200system (Xenogen Corp.). Alternatively, vvDD-GFP was used (lower panelday 3) in an equivalent experiment, and animals were imaged using aMaestro (CRI, Boston Mass.) imaging system. (b) Quantification of lightoutput per tumor is plotted relative to time 13 as an indication ofviral replication and distribution. Values are averages for 3 animalsper group, error bars are SEM; p=0.0079 at day 35 (T-test). (c) Tumorsfrom animals receiving vvDD-GFP (1×10₇ PFU) or 1×10₇ CIK pre-infectedwith vvDD-GFP (MOI 1.0 PFU/cell) via tail vein injection. Sections (48 hpost-injection) were stained with anti-CD31 (endothelial cell marker)(magnification 400×) (d) Tumor from animals receiving 1×10₇ CIK cellsconjugated to Cy5.5 (red) and pre-infected with vvDD-GFP (MOI 1.0PFU/cell). Sections were stained with Sytox blue (DNA binding)(blue) andanti-GFP antibody (green) (tumors taken 72 h post-injection). Arrowsindicate area of overlapping green and red, indicating infected CIKcells within the tumor (scale bar 100

m).

FIG. 4A-4B. Survival and tumor burden of animals bearing UCI-101 orSKOV-3 tumors. (a) Kaplan-Meier survival curves of animals carryingUCI-101 or SKOV-3 intraperitoneal tumors (cell lines expressedluciferase and tumor burden was measured using bioluminescence imaging).Each animal received a single tail vein injection of either PBS; 1×10₇CIK cells; 1×10₇ PFU vvDD; or 1×10₇ CIK cells preinfected with vvDD (MOI1.0 PFU/cell), n=8 animals/group. Combination therapy significantlyincreased survival compared to any other treatment (Logrank test;p=<0.05). (e.g. CIK+vvDD compared to vvDD alone; p=0.0072 (UCI-101) or0.0379 (SKOV-3)). (b) The tumor burden (measured by bioluminescenceimaging) for each individual animal was plotted against time. A singleintravenous treatment was delivered on day 3 (arrows) of PBS (black) orcombination therapy (green) (top) or CIK (red) or vvDD (blue) (bottom).Grey shaded area indicates range of tumor burden for the PBS treatedgroup.

FIG. 5A-5B. Fluoresence and bioluminescence imaging of trafficking ofuninfected and vvDD infected CIK cells. (a) Mice carrying subcutaneousSKOV-3 (top panel) or UCI-101 (bottom panel) tumors were treated (day 0)with a single intravenous injection of either 1×10₇ CIK cells labeledwith Cy5.5 (left animal) or 1×10₇ Cy5.5 labeled CIK cells infected withvvDD-GFP (MOI 1.0) (right animal). Cy5.5 fluorescence was imaged usingan IVIS200 system (Xenogen Corp.). A PBS control mouse was included 14for comparison on day 2. (b) In an equivalent experiment, a mousebearing an UCI-101 tumor was treated with 1×10₇ CIK cells transfectedwith retrovirus to express luciferase and infected with vvDD-GFP (MOI1.0) on day 0. Bioluminescence was imaged using an IVIS200. Arrowsindicate locations of tumors.

FIG. 6A-6E. Immunofluorescence microscopy of MICA/B expression in atreated SKOV-3 tumor. A mouse bearing a subcutaneous SKOV-3 tumor wastreated with a tail vein injection of 1×10₇ CIK cells labeled with Cy5.5and infected with vvDD-GFP (MOI 1.0). Tumors were recovered 48 hoursafter treatment and frozen sections were stained with (a) a nuclear dye(Sytox Blue, Molecular Probes); (b) anti-GFP antibody: or (c)anti-MICA/B. Fluorescence was imaged, along with Cy5.5 fluorescence (d)using a Leica confocal microscope. An overlay image is also shown (e)

FIG. 7. Immunofluorescence microscopy of CIK delivery of vvDD in aUCI-101 tumor. A mouse bearing a subcutaneous UCI-101 tumor was treatedwith a tail vein injection of 1×10₇ CIK cells labeled with Cy5.5 andinfected with vvDD-GFP (MOI 1.0). Tumors were recovered 24 hours aftertreatment and frozen sections were stained with a nuclear dye (SytoxBlue, Molecular Probes) and anti-GFP antibody (green). Fluorescence wasimaged, along with Cy5.5 fluorescence (red) using a Leica confocalmicroscope. An overalay image is shown>Style tag for figure legends.

FIG. 8. Graphs depicting the relapse of lymphoma growth in mice treatedwith PBS alone; CIK cells alone; and CIK cells pre-infected withvaccinia virus.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The objects and advantages of the present invention are achieved by amethod of treating malignancy in a patient; comprising administering aneffective amount of immune effector cells pre-infected with an oncolyticvirus, to a patient afflicted with cancer. Oncolytic viruses of interestare replication-selective or tropism modified viruses that are eitheronly capable of entering into, or of completing a successful infectioncycle within, transformed cells.

It has been found that a surprising synergy is obtained by infectingimmune effector cells with an oncolytic virus. The infection of thetumor cells sensitizes them to subsequent effector cell-mediatedkilling. Without limiting the scope of the invention, it is believedthat the effect may be mediated by expression of proteins on the cellsurface that are associated with cellular stress, and which enhanceeffector cell killing. Further, virus delivered via infected immuneeffector cells produced a more uniform biodistribution of infectionwithin the tumor, even at locations distant to the tumor vasculature.The combination therapy was capable of producing dramatically increasedsurvival compared to either therapy alone.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed herein are standard methodologies well known to oneof ordinary skill in the art.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994); and “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991).

As used herein, a “target cell” is a tumor cell in which the virusreplicates. Usually a target cell is a mammalian cell, preferably ahuman cell.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient of any virus and/or virus vector(s) of thisinvention. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or oftotal DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation and/or change. A host cell includescells transfected or infected in vivo with a polynucleotide and/or avector of this invention.

As used herein, the terms “neoplastic cells”, “neoplasia”,“transformed”, “tumor”, “tumor cells”, “cancer” and “cancer cells”,(used interchangeably) refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation.Neoplastic cells can be malignant or benign.

The term “gene” is well understood in the art and is a polynucleotideencoding a polypeptide. In addition to the polypeptide coding regions, agene includes non-coding regions including, but not limited to, introns,transcribed but untranslated segments, and regulatory elements upstreamand downstream of the coding segments.

“Replication” and “propagation” are used interchangeably and refer tothe ability of a virus to reproduce or proliferate. This term is wellunderstood in the art. For purposes of this invention, replicationinvolves production of virus proteins and is generally directed toreplication of the virus genome. Replication can be measured usingassays standard in the art and described herein, such as a burst assayor plaque assay. “Replication” and “propagation” include any activitydirectly or indirectly involved in the process of virus manufacture,including, but not limited to, viral gene expression, production ofviral proteins, nucleic acids or other components, packaging of viralcomponents into complete viruses, and cell lysis.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length. Theseterms also include proteins that are post-translationally modifiedthrough reactions that include glycosylation, acetylation andphosphorylation.

As used herein, “cytotoxicity” is a term well understood in the art andrefers to a state in which one or more of a cell's usual biochemical orbiological functions are perturbed. These activities include, but arenot limited to, metabolism, cellular replication, DNA replication,transcription, translation, and uptake of molecules. “Cytotoxicity”includes cell death and/or cytolysis. Assays are known in the art whichindicate cytotoxicity, such as dye exclusion, ³H-thymidine uptake, andplaque assays. The term “selective cytotoxicity”, as used herein, refersto the cytotoxicity conferred by a virus on a target cell, compared tothe cytotoxicity conferred by an virus on a non-permissive cell. Suchcytoxicity may be measured, for example, by plaque assays, reduction orstabilization in size of a tumor comprising target cells, or thereduction or stabilization of serum levels of a marker characteristic ofthe tumor cells or a tissue-specific marker, e.g., a cancer marker suchas prostate specific antigen.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, rodents, primates, farmanimals, sport animals, and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. For purposes of this invention, an effectiveamount of the therapy is an amount that is sufficient to palliate,ameliorate, stabilize, reverse, slow or delay the progression of thedisease state.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, preventing spread (i.e., metastasis) ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. “Palliating” a disease means that the extent and/orundesirable clinical manifestations of a disease state are lessenedand/or time course of the progression is slowed or lengthened, ascompared to not administering vectors of the present invention.

Oncolytic virus. Oncolytic viruses are viruses, which, when brought intocontact with tumor cells, are capable of killing those cells. Viruses ofinterest are replication-selective or tropism modified for tumor cells,and often have been genetically modified to reduce replicationcapability in non-transformed cells. Viruses of interest includeadenovirus; herpes simplex virus-1; vaccinia virus; parvovirus;reovirus; Newcastle disease virus; and the like. Vaccinia virus is ofparticular interest.

As used herein, “virus” refers to the virus itself or derivativesthereof. The term covers all serotypes and subtypes and both naturallyoccurring and recombinant forms, except where otherwise indicated. Anviral vector of the present invention can be in any of several forms,including, but not limited to, naked viral genomic DNA; a viral genomeencapsulated in an virus coat; packaged in another viral or viral-likeform (such as herpes simplex virus and AAV); encapsulated in a liposome;complexed with polylysine or other biocompatible polymer; complexed withsynthetic polycationic molecules; conjugated with transferrin; complexedwith compounds such as PEG to immunologically “mask” the molecule and/orincrease half-life, or conjugated to a non-viral protein. For purposesof this invention, virus vectors are replication-competent in a targettumor cell.

Mechanistic approaches to tumor-selective replication include the use ofviruses with inherent tumor selectivity (for example, Newcastle diseasevirus (NDV), reovirus, vesicular stomatitis virus (VSV) and autonomousparvovirus); deletion of entire genes (herpes simplex virus (HSV),adenovirus and vaccinia virus) or functional gene regions (adenovirusand poliovirus) that are necessary for efficient replication and/ortoxicity in normal cells but are expendable in tumor cells; engineeringof tumor/tissue-specific promoters into viruses to limit expression ofgene(s) necessary for replication to cancer cells (adenovirus and HSV);and modification of the viral coat to selectively target uptake to tumorcells (adenovirus and poliovirus).

Specific modifications known in the art include modifications to thevirus for tumor selectivity include deletion of viral genes necessaryfor replication in normal cells but not tumor cells, e.g. adenovirus E1B55 kD deletion; HSV-1 ribonucleotide reductase subunit disruption;adenovirus E1A CR1 or CR2 deletion; poliovirus PV1 deletion; etc. Tumorspecific promoter element may be used to drive expression of early viralgenes, e.g. adenovirus E1A under the control of alpha fetoproteinpromoter/enhancer; adenovirus E1A or E1B under the control of PSApromoter/enhancer; adenovirus E1A under the control of alpha fetoproteinpromoter/enhancer; HSV-1 ICP4 gene under the control of albuminpromoter/enhancer; etc. The virus may be engineered to express atumor-selective receptor in the virus coat, e.g. adenovirus having areplacement of CAR/integrin-binding with a tumor targeting ligand; andthe like.

The oncolytic virus may further comprise a coding sequence or codingsequences for a protein or proteins to be administered to the patient.In some embodiments, such proteins are those that are cytotoxic orcytostatic for tumor cells, e.g. tumor necrosis factor (TNF),interleukin-2 (IL-2), drugs that can be utilized in an effectivechemotherapeutic regime, such as multiple drug resistant protein. TheDNA encoding such active protein ingredient may be obtained from anyconvenient source and, depending on the protein chosen, can besynthesized chemically, recovered from a cDNA library, isolated fromgenomic DNA, or otherwise obtained by means known in the art.

Proteins of interest include any protein(s) which has a desired effecton an infected cell in the subject to be treated. Advantages of the drugdelivery system of the invention are experienced especially when theprotein operates within the cytoplasm of a target cell or is releasedfrom an infected or lysed cell to induce a cytotoxic or cytostaticeffect on neighboring cells (bystander effect). For example, tumornecrosis factor (TNF) is capable of selectively killing tumor cells, butneeds to transit the cell membrane to exert its effect. Other proteins,such as ribotoxins and the various colony-stimulating factors, alsooperate intracellularly.

It will be appreciated that viruses constructed to express two genes mayinclude two proteins that have prophylactic or therapeutic value, or onegene could express a dominate selectable marker which would facilitateidentifying cells transfected with the two gene construct. An example ofa selectable marker would be resistance to G418 that is conferred oncells by the presence of the neomycin gene sequences. Alternatively asecond gene may be used for imaging purposes, for example the SodiumIodide symporter (NIS) gene or somatostatin receptor gene may beexpressed in order to locate and quantify viral gene expression bydelivery of a radiolabelled tracer followed by PET or SPECT imaging.

DNAs encoding the foregoing proteins are available in the art, and canbe obtained bracketed with linker sequences for convenient manipulation,if desired. The nature of the delivery system is such that both genomicand cDNA sequences can be used, since introns can be processed in theenvironment transfected by the provirus. The protein drug can be encodedin the delivery virion to specify any form of the protein desired, forexample, an active form, a mature form, a fused protein, a preprotein,or a preproprotein.

Vaccinia virus is of particular interest for the methods of theinvention. Wild-type vaccinia virus is well tolerated following bothintratumoral and intravesical treatment, and vaccinia viruses expressingtumor-associated antigens or proinflammatory cytokines have been welltolerated in a number of Phase I trials using subcutaneous, intradermalor intratumoral inoculation. Vaccinia, unlike most viruses proposed foruse in virotherapy, has the advantage of some demonstrated systemicdelivery potential. However, since the virus is capable of infectingmany different cell types, only a small fraction of the incoculum of anystrain used for virotherapy may reach the tumor. Therefore, developmentof an effective means of delivering viruses to tumor targets isnecessary for the development of effective therapy.

In one embodiment of the invention, the oncolytic virus is a vacciniavirus. All vaccinia strains tested, including the wild-type strain, showunusual replication kinetics in immune effector cells, where there is aninitial extended eclipse period of slow replication followed by a rapidburst of replication. The eclipse phase is at least about 1 day, usuallyat least about 2 days, and not more than about 4 days, usually after notmore than about 3 days post-infection.

A number of vaccinia virus strains have been shown to display an eclipsephase in CIK cells, and may be used in the methods of the invention. Thevaccinia strain Western Reserve (WR) alone or carrying deletions in theTK, VGF or both genes displayed an eclipse phase, which eclipse phasewas ‘enhanced’ in the double deleted virus. Other vaccinia strains,including Wyeth and International Health Department (IHD) also displayedeclipse phases in CIK cells. In addition a variety of deletion mutationsin the WR backbone also displayed an eclipse phase, of note deletionsthat enhanced the production of the EEV (Extracellular Enveloped Virus)form of the virus and deletions that prevented the production ofextracellular IFN binding proteins.

Replication of vaccinia containing a deletion of the thymidine kinase(TK) gene has been shown to be restricted to cells with elevatedcellular levels of thymidine kinase, e.g. dividing cells and tumorcells. Vaccinia with TK deletions will replicate both in the effectorcell cultures, where cell division has been stimulated by anti-CD3antibody, and in tumor cells. Thus, in one embodiment, the oncolyticvirus is a vaccinia virus comprising a genetic modification thatsubstantially eliminates active TK expressed from the viral genome.

Viral genes whose deletion does not effect, or enhances eclipse phaseinclude, without limitation, TK, VGF, B5R (EEV producing); B8R (IFNγbinding protein); B18R (Type I IFN binding protein). The oncolytic virusmay comprise a genetic modification that substantially eliminates activeviral growth factor (VGF); B5R (EEV producing); B8R (IFNγ bindingprotein); or B18R (Type I IFN binding protein) expressed from the viralgenome. For example, the VGF gene product promotes cellular growth aftersecretion from infected cells, by interacting with growth factorreceptors. VGF deletions have been shown to restrict viral replicationto cells with mutations in the Ras/MAPK/ERK pathway, offering additionaltumor selectivity. In one embodiment, the oncolytic virus is a vacciniavirus comprising a genetic modification that substantially eliminatesactive TK expressed from the viral genome and active VGF expressed fromthe viral genome.

“Genetic alteration”, or “mutation”, to decrease expression of genes ofinterest as described above, refers to any alteration to a gene whereinthe expression of that gene is significantly decreased, or wherein thegene product is rendered nonfunctional, or its ability to function issignificantly decreased. The term “gene” encompasses both the regionscoding the gene product as well as regulatory regions for that gene,such as a promoter or enhancer. Such alterations render the product ofthe gene non-functional or reduce the expression of the gene such thatthe viral mutant has the properties of the instant invention. Moreover,the invention encompasses mutants with one or more mutation(s) in one ormore gene(s) of interest. Thus, by “a” is intended one or more. Forexample, “a mutation in a TK gene” means that there can be one or moremutations in one or more TK genes.

Ways to achieve such alterations include any method to disrupt theexpression of the product of the gene or any method to render theexpressed protein nonfunctional. Numerous methods known to disrupt theexpression of a gene are known, including the alterations of the codingregion of the gene, or its promoter sequence in the by insertions,deletions and/or base changes. (See, Roizman and Jenkins, Science 229:1208 (1985)).

A preferred mutation is the deletion of nucleic acids from a gene.Methods for the construction of engineered viruses and for the geneticmanipulation of DNA sequences are known in the art. Generally, theseinclude Ausubel et al., Chapter 16 in Current Protocols in MolecularBiology (John Wiley and Sons, Inc.); Paoletti et al., U.S. Pat. No.4,603,112 (July 1986). Virological considerations also are reviewed inCoen, in Virology, 1990 (2.sup.nd ed.) Raven Press, pages 123-150.

Immune Effector cells. Immune effector cells, for the purposes of theinvention, are autologous or allogeneic immune cells having cytolyticactivity against tumor cells. The effector cells may have cytolyticactivity that does not require recognition through the T cell antigenreceptor. Cells of particular interest include cells of the T and/or NKlineage, e.g. LAK cells, CIK cells, CTL, TIL cells, etc. The effectorcells are typically obtained by culturing peripheral blood lymphocytes(PBL) in vitro with a cytokine and/or antigen combination that increasesactivation. In the methods of the invention, the activated effectorcells are infected with virus, and administered to the patient. Thecells are optionally separated from non-desired cells prior to culture,prior to administration, or both. Cell-mediated cytolysis of tumor cellsby immunological effector cells is believed to be mediated by the localdirected exocytosis of cytoplasmic granules that penetrate the cellmembrane of the bound target cell.

Natural killer (NK) cells are cytotoxic cells belonging to a cell classresponsible for cellular cytotoxicity without prior sensitization.IL-2-activated NK cells, the major effector population inlymphokine-activated killer (LAK) cells, are potent mediators of thelysis of autologous and allogeneic leukemic cells in vitro. LAK cellsare non-B, non-T cells that are capable of recognizing cancer cells in anon-MHC-restricted fashion. LAK cells, which can be generated fromeither the normal or tumor-bearing host, appeared to represent aprimitive immunosurveillance system capable of recognizing anddestroying altered cells. NK cells often do not react with patient tumorcells unless they are activated by interferon, IL-2, or unlesssuppressor monocytes are removed from the effector cell population. IL-2induces proliferation of T lymphocytes and NK cells and the productionof IFN-gamma; it also results in the induction of LAK cells againstpreviously NK-resistant cell preparations and cell lines. LAK activitycan be generated from human and murine T cells following incubation withIL-2.

Most NK activity, as well as LAK activity, is mediated by the CD3⁻ largegranular lymphocyte (LGL) cell population. This lytic activity isobserved against a variety of tumor cells and virally infected cells.Morphologically, NK cells are characterized as LGL cells containing akidney-shaped nucleus and prominent azurophilic granules in theircytoplasm. Human LGL share both myelomonocytic (e.g., CD11) and T cell(e.g., CD2 and CD8)-related markers. However, the majority of human NKcell activity is mediated by CD3⁻, CD56⁺, and CD16⁺ lymphocytes,although CD16⁻ NK cells have also been characterized. The CD16c cellsalso have high levels of antibody-dependent cellular cytotoxicity(ADCC). The CD16⁻ NK cells express markers similar to CD16⁺ NK cells,including CD2, CD7, CD11b, CD38, CD45R, CD18, and p75 IL-2R. LAK cellshave been utilized in vivo both in animals and in human beings for thetreatment of melanoma, renal cell carcinoma, non-Hodgkin's lymphoma, andlung and colorectal cancers.

Cytotoxic T lymphocytes (CTL) reactive to autologous tumor cells arespecific effector cells for adoptive immunotherapy. Induction andexpansion of CTL is antigen-specific, and MHC restricted. Various typesof cytokines other than IL-2 have also been reported to induce cytotoxiclymphocytes. A class of T lymphocytes with antitumor activity has beentermed “tumor-infiltrating lymphocytes” (TIL). They possess more potentantitumor activity than LAK cells. They can be grown by culturingsingle-cell suspensions obtained from tumors in IL-2. Althoughlymphocytes comprise only a small subpopulation of the cells in a cancernodule, some of these lymphocytes contain IL-2 receptors and grow underthe influence of IL-2. Although tumor cells also grow in the culture,lymphocytes capable of eliminating the tumor cells have a selectivegrowth advantage. After 2-3 weeks of culture, pure populations oflymphocytes without contaminating tumor cells are obtained. The majoreffectors of TIL cells are phenotypically CD3⁺CD56⁻CD⁸⁺ and are MHCrestricted. Cancer patients have been treated with ex vivoanti-CD3-activated killer cells and IL-2.

Cytokine-induced killer (CIK) cells are highly efficient cytotoxiceffector cells obtained by culturing peripheral blood lymphocytes (PBLs)in the presence of IFN-gamma, IL-2 (or IL-12), and monoclonal antibody(MAb) against CD3, and optionally include IL-1a. Cells may be culturedfor at least about 1 week, at least about 2 week, at least about 3weeks, or more, and usually not more than about 8 weeks in culture. Theabsolute number of CIK effector cells usually increases at least about100-fold in such culture conditions, and may increase at least about500-fold, at least about 1000-fold, or more.

CIK cells possess a higher level of cytotoxic activity and a higherproliferation rate than LAK cells. The phenotype of the cells with thegreatest cytotoxicity expresses both the T-cell marker CD3 and the NKcell marker CD56. The expression of CD56 by the antigen is correlatedwith antitumor cytotoxicity. CD28, a major co-stimulatory signal of theTCR, is present on the cell surface only in a subset of CIK cells. CIKcells secrete IL-2, IL-6 and TNF-alpha. IL4, IL-7, and IL-12 are notsecreted. Most of these CD3⁺CD33⁺ cells co-expressed CD2, CD5, CD7, CD8and HLA-DR but were negative for expression of CD4, CD13, CD14, CD15,and CD16. CD3cCD33c cells possessed no cytotoxic activity against tumorcells. CD3⁺CD56⁺ cells are expanded dramatically in CIK cell cultures.The percentage of CD3⁺CD56⁺ cells reaches a plateau after approximately1-2 months of culture. The dominant cell phenotype in CIK cell culturesexpressed the alpha-, beta-T-cell receptor (TCR-α/β). In comparison toNK cells, the cytotoxicity mediated by CD3⁺CD56⁺ cells is also non-MHCrestricted in the absence of activation, but it is non-ADCC dependent,since these double-positive cells do not express CD16. Morphologically,these cells cannot be distinguished from NK cells.

A potent in vivo antitumor effect of CIK cells in an animal model can beachieved with as few as 1×10⁷ cells. In humans, an effective dose isusually at least about 10⁸ cells, at least about 10⁹ cells, and may be10¹⁰ cells, or more.

Immune effector cells useful in the methods of the invention have an“eclipse” period following infection with the oncolytic virus. It hasbeen found that cells possessing this property include, withoutlimitation, T cell lines and CIK cells, where vaccinia displayed a cleareclipse phase in these cells in culture.

Cancer, as used herein, refers to hyperproliferative conditions. Theterm usually denotes malignant cell populations. Such disorders have anexcess cell proliferation of one or more subsets of cells, which oftenappear to differ from the surrounding tissue both morphologically andgenotypically. The excess cell proliferation can be determined byreference to the general population and/or by reference to a particularpatient, e.g. at an earlier point in the patient's life.Hyperproliferative cell disorders can occur in different types ofanimals and in humans, and produce different physical manifestationsdepending upon the affected cells.

Cancers include leukemias, lymphomas (Hodgkins and non-Hodgkins),sarcomas, melanomas, adenomas, carcinomas of solid tissue includingbreast cancer and pancreatic cancer, hypoxic tumors, squamous cellcarcinomas of the mouth, throat, larynx, and lung, genitourinary cancerssuch as cervical and bladder cancer, hematopoietic cancers, head andneck cancers, and nervous system cancers, such as gliomas, astrocytomas,meningiomas, etc., benign lesions such as papillomas, and the like.

Cancers of interest include, but are not limited to, ovarian and breastcancer and lymphomas. Ovarian cancer is the second most commonlydiagnosed gynecologic malignancy, the deadliest gynecologic malignancy,and the fourth leading cause of cancer-related deaths in women in theUSA. About 1 in 70 women eventually develops ovarian cancer, and 1 in100 women dies of it. Ovarian cancer affects predominantlyperimenopausal and postmenopausal women.

Ovarian tumors are the most histologically diverse group of tumors. Atleast 80% of malignant ovarian tumors arise from the coelomicepithelium. The most common type is serous cystadenocarcinoma, whichaccounts for 75% of cases of epithelial ovarian cancer. Others includemucinous, endometroid, transitional cell, Brenner, clear cell, andunclassified carcinomas. The remaining 20% of malignant ovarian tumorsare germ cell and sex cord-stromal cell tumors, which are nonepithelialin origin, and metastatic carcinomas to the ovary (most commonly, breastand GI carcinomas). Germ cell tumors, which arise from the primary germcells of the ovary, occur in young women and are uncommon in women >30yr. Malignant germ cell tumors include dysgerminomas, immatureteratomas, endodermal sinus tumors, embryonal carcinomas,choriocarcinoma, and polyembryomas. Stromal malignancies includegranulosa-theca cell tumors and Sertoli-Leydig cell tumors.

Ovarian cancer spreads by direct extension, by intraperitonealimplantation via exfoliation of cells into the peritoneal cavity, bylymphatic dissemination in the pelvis and para-aortic region, and, lesscommonly, hematogenously to the liver or lungs.

CA 125 is a cell surface glycoprotein detectable in 80% of cases ofepithelial ovarian cancer. However, it is not specific to patients withovarian cancer and, among premenopausal patients, can be mildly elevatedin several benign disorders, including endometriosis, pelvicinflammatory disease, pregnancy, and leiomyomata uteri.

For patients with advanced-stage epithelial ovarian cancer,cytoreductive (tumor-debulking) surgery is advised to improve theefficacy of adjunctive therapies. The goal is to reduce the tumor burdenso that the maximum diameter of the remaining implants is <1 cm.Cytoreductive surgery usually includes total hysterectomy, bilateralsalpingo-oophorectomy, omentectomy, and excision of the tumor from anyother sites. Rectosigmoid resection (usually with primaryreanastomosis), radical peritoneal stripping, resection of diaphragmaticperitoneum, or splenectomy may be required. Prognosis for patients withadvanced disease is directly related to the success of cytoreductivesurgery.

Methods of Treatment

The methods of the invention involve the culture of immune effectorcells in vitro, the infection of the immune effector cells with anoncolytic virus, and the administration of the infected cells to patientsuffering from cancer. The treatment is intended to reduce or eliminatecancer in the patient. Also provided are compositions of immune effectorcells infected with an oncolytic virus, which cells may be provided in aculture medium, in aliquots suitable fro delivery to a patient, and thelike.

The immune effector cells are usually generated from a fresh or frozenhematopoietic cell population. The source of cells may be autologous orallogeneic relative to the patient, but will usually be of the samespecies, e.g. human cells for human patients, mouse cells for a mouserecipient, etc. Cell populations include, but are not limited to, cellpopulations obtained from peripheral blood, spleen, lymph node, bonemarrow, mobilized peripheral blood, umbilical cord blood, etc.

Procedures for separation can include, but are not limited to, physicalseparation, magnetic separation, using antibody-coated magnetic beads,affinity chromatography, cytotoxic agents joined to a monoclonalantibody or used in conjunction with a monoclonal antibody, including,but not limited to, complement and cytotoxins, and “panning” withantibody attached to a solid matrix, e.g., plate, elutriation or anyother convenient technique. The use of physical separation techniquesinclude, but are not limited to, those based on differences in physical(density gradient centrifugation and counter-flow centrifugalelutriation), cell surface (lectin and antibody affinity), and vitalstaining properties (mitochondria-binding dye rho123 and DNA-binding dyeHoechst 33342). These procedures are well known to those of skill inthis art.

The cells are cultured as described herein to provide for an activatedcell population. Such culture typically reduces the number ofnon-desired cells, e.g. alloreactive T cells, etc. Undesired cells mayalso be removed by selection. Separation of the desired cells forengraftment will generally use affinity separation. Techniques providingaccurate separation include fluorescence activated cell sorters, whichcan have varying degrees of sophistication, such as multiple colorchannels, low angle and obtuse light scattering detecting channels,impedance channels, etc. The cells may be selected against dead cells byemploying dyes associated with dead cells (propidium iodide, LDS). Anytechnique may be employed which is not unduly detrimental to theviability of the selected cells.

The affinity reagents may be specific receptors or ligands for cellsurface molecules, e.g. CD8, CD4, etc. In addition to antibody reagents,peptide-MHC antigen and T cell receptor pairs may be used; peptideligands and receptor; ligand and receptor molecules, and the like.Antibodies and T cell receptors may be monoclonal or polyclonal, and maybe produced by transgenic animals, immunized animals, immortalized humanor animal B-cells, cells transfected with DNA vectors encoding theantibody or T cell receptor, etc. The details of the preparation ofantibodies and their suitability for use as specific binding agents arewell-known to those skilled in the art.

The specific culture condition will be selected based on the type ofeffector cell that is desired. Where the effector cells are CIK cells,for example, the cells are washed and resuspended at in medium, e.g.RPMI, DME, etc. Interferon γ is added to culture at an effectiveconcentration, e.g. at from about 100 to about 10,000 U/ml, usuallyaround about 1000 U/ml. A ligand specific for CD3, e.g. monoclonalanti-CD3 antibody is added to the culture at an effective concentration,for example OKT3 may be used at a concentration of from about 1 to about500 ng/ml. The medium is changed at IL-2 added at regular intervals. Theeffector cells are preferably taken when the CIK population hasexpanded.

The population of effector cells is infected with the cytolytic virus.Preferably the virus does not cause cytolysis of the effector cells inthe period of time between infection and patient administration. Asdescribed herein, an eclipse period of from about 1 day to not more thanabout 4 days provides a window of time where the virus does not causesignificant cytolysis of the effector cells.

If used as a packaged virus, the virus may be administered in anappropriate physiologically acceptable carrier. The multiplicity ofinfection will generally be in the range of about 0.001 to 100. Theviruses may be administered one or more times.

Alternatively, viral DNA may be used to transfect the effector cells,employing liposomes, general transfection methods that are well known inthe art (such as calcium phosphate precipitation and electroporation),etc. Due to the high efficiency of transfection of viruses, one canachieve a high level of modified cells.

The population of infected effector cells are injected into therecipient. Determination of suitability of administering cells of theinvention will depend, inter alia, on assessable clinical parameterssuch as serological indications and histological examination of tissuebiopsies. Generally, a pharmaceutical composition is administered.

Routes of administration include systemic injection, e.g. intravascular,subcutaneous, or intraperitoneal injection, intratumor injection, etc.Where the recipient animal is a human, the number of cells injected willusually be at least about 0.5×10⁸ and not more than about 5×10¹⁰, moreusually at least about 1×10⁸ or at least about 1×10⁹.

The invention provides methods of suppressing tumor cell growth,comprising contacting a tumor cell with an infected effector cell of theinvention such that the oncolytic virus enters the tumor cell, and thereis selective cytotoxicity for the tumor cell. The composition may beadministered once, or a series of times, e.g. daily, weekly,semi-monthly, etc. The efficacy may be monitored by standard methods asappropriate to the specific cancer, e.g. tumor size, biopsy, presence oftumor cells in the blood, etc. Tumor cell growth can be assessed bydetermining whether tumor cells are proliferating using a ³H-thymidineincorporation assay, or counting tumor cells. “Suppressing” tumor cellgrowth means any or all of the following states: slowing, delaying, andstopping tumor growth, as well as tumor shrinkage. “Suppressing” tumorgrowth indicates a growth state that is curtailed when compared togrowth without contact with the cells of the invention.

The present invention also includes compositions, includingpharmaceutical compositions, containing the infected effector cellsdescribed herein. Such compositions are useful for administration, forexample, when measuring the effectiveness of cell killing in anindividual. Preferably, these compositions further comprise apharmaceutically acceptable excipient. These compositions, which cancomprise an effective amount of an the infected effector cells of thisinvention in a pharmaceutically acceptable excipient, are suitable forsystemic administration to individuals in unit dosage forms, sterileparenteral solutions or suspensions, sterile non-parenteral solutions ororal solutions or suspensions, oil in water or water in oil emulsionsand the like. Formulations for parenteral and nonparenteral drugdelivery are known in the art and are set forth in Remington'sPharmaceutical Sciences, 18.sup.th Edition, Mack Publishing (1990).Compositions also include lyophilized and/or reconstituted forms of thevectors of the invention.

The methods of the combination may be combined with conventionalchemotherapeutic, radiologic and/or surgical methods of treatment.Cytotoxic agents that act to reduce cellular proliferation are known inthe art and widely used. Such agents include alkylating agents, such asnitrogen mustards, e.g. mechlorethamine, cyclophosphamide, melphalan(L-sarcolysin), etc.; and nitrosoureas, e.g. carmustine (BCNU),lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin,etc. Antimetabolite agents include pyrimidines, e.g. cytarabine(CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine(FUdR), etc.; purines, e.g. thioguanine (6-thioguanine), mercaptopurine(6-MP), pentostatin, fluorouracil (5-FU) etc.; and folic acid analogs,e.g. methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, etc. Other naturalproducts include azathioprine; brequinar; alkaloids and synthetic orsemi-synthetic derivatives thereof, e.g. vincristine, vinblastine,vinorelbine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithromycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; and the like.Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine. Other anti-proliferative agents of interestinclude immunosuppressants, e.g. mycophenolic acid, thalidomide,desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF105685), etc. The antineoplastic agents taxols (or taxanes)hyperstabilize polymerized microtubules, leading to mitotic arrest andcytotoxicity in proliferating cells. Taxanes (or taxols), such aspaclitaxel, docetaxel, etc. are of interest. Also of interest are themicrotubule stabilizing epothilones (see Bollag et al. (1995) CancerResearch, Vol 55, Issue 11 2325-2333, herein incorporated by referencewith respect to teachings of the class, and use thereof of thesechemotherapeutic agents), e.g. epothilone A and epothilone B. Retinoids,e.g. vitamin A, 13-cis-retinoic acid, trans-retinoic acid, isotretinoin,etc.; carotenoids, e.g. beta-carotene, vitamin D, etc. Retinoidsregulate epithelial cell differentiation and proliferation, and are usedin both treatment and prophylaxis of epithelial hyperproliferativedisorders.

The present invention relates to the selective killing of neoplasticcells by combined viral mediated oncolysis and immune effector therapy,which combination provides a synergistic benefit when compared to eitherof the single therapies. The invention provides for oncolytic virusinfected immune effector cells, a method of killing neoplastic cellsusing oncolytic virus infected immune effector cells, and apharmaceutical composition containing oncolytic virus infected immuneeffector cells. In the combined therapeutic, the effector cells wereshown to retain their ability to traffic to tumors. At the tumor sitethe oncolytic virus was released deep in the tumor rather than merely atthe surface; thus the cell mediated delivery of the virus led toenhanced biodistribution within the tumor. In addition, the cytotoxiceffects of the effector cells may be increased by viral replication inthe tumor target. This combined therapeutic has been demonstrated to besafe, with minimal viral infection of normal tissues, and highlyeffective.

It has been shown that the effector cells of the invention can be usedto deliver oncolytic virus to a recipient capable of mounting ananamnestic response against the oncolytic virus, e.g. a vaccinia viruscan be successfully delivered to a patient previously immunized withvaccinia. This property overcomes a limitation of previously describedviral therapy, where a single agent, e.g. an oncolytic virus, isineffective in repeat doses or in vaccinated individuals due to therecipient immune response. It has been found that a polyclonal,anti-vaccinia antibody failed to recognize CIK cells infected withvaccinia, although the antibody did recognize tumor cells infected withvaccinia. VIG (vaccinia immunoglobulin) is an FDA approved treatment foradverse events following smallpox vaccination. Doses of VIG thatcompletely neutralized vaccinia (prevented vaccinia from infecting acell layer following 2 h of exposure to VIG) had no effect on theability of infected CIK cells to transfer virus to a cell layer. Animalstreated with vaccinia (or infected CIK cells) produced CTLs (cytotoxic Tlymphocytes) that could recognize infected tumor cells, but not infectedCIK cells. Tumor bearing and immunized mice were treated (IV) withvaccinia or infected CIK cells. There was very little evidence ofvaccinia in the tumor when it was delivered alone. There were multipleareas of positively stained cells (for vaccinia infection) within thetumors when infected CIK cells were used. Tumor bearing animals treatedwith high doses (10× therapeutic levels) of VIG, and then treated withinfected CIK cells still produced signal (bioluminescence imaging fromvirally encoded luciferase) from within the tumor.

EXPERIMENTAL Example 1

Targeted biological therapies hold tremendous potential for thetreatment of cancers, yet their effective use has been limited byconstraints on delivery and effective tumor targeting. Here we combinean immune effector cell population; cytokine induced killer (CIK) cells,with a complementary oncolytic viral therapy as an effective treatmentof ovarian cancer. CIK cells, an ex vivo expanded population of cellsderived from human peripheral blood, were used to deliver a replicationcompetent vaccinia virus carrying genetic deletions that restrict itsreplication to malignant cells. Pre-infection of CIK cells withoncolytic vaccinia virus resulted in a prolonged eclipse period wherethe virus remained within the CIK cells until interaction with, andinfiltration into, the tumor. In this combined therapeutic not only didthe CIK cells retain their ability to traffic to ovarian tumors, butonce at the tumor site the oncolytic virus was released deep in thetumor rather than merely at the surface; cell mediated delivery of thevirus led to enhanced biodistribution within the tumor. In addition, thecytotoxic effects of the CIK cells were increased by viral replicationin the tumor target. This combined therapeutic was both safe, withminimal viral infection of normal tissues, and highly effective,producing a dramatically enhanced anti-tumor effect compared to eithertherapy alone. This effective new biological approach for the treatmentof cancer, demonstrates that synergistic effects of combinedbiotherapeutics can be used to effectively kill difficult-to-treatcancers.

Viral replication kinetics were determined with selected vacciniastrains in CIK cells and compared to the parental strain (WesternReserve, WR). All vaccinia strains (including WR) displayed unusualreplication kinetics in CIK cells (FIG. 1 a) compared to the very rapidand lytic replication seen in other cell lines such as the lymphomaOCI-ly8 (FIG. 1 b). There appeared to be a two-step growth curve in theCIK cells, with an initial extended eclipse period of slow replicationfollowed by a rapid burst of replication between 48 and 72 hpost-infection. Replication of vaccinia containing a deletion of thethymidine kinase (TK) gene has been shown to be restricted to cells withelevated cellular levels of thymidine kinase as observed in the G2- andS-phase of the cell cycle of normal cells. In cancer cells TK activityis constitutively high, and therefore vaccinia with TK deletions shouldreplicate both in the CIK cultures, where cell division has beenstimulated by anti-CD3 antibody, and in tumor cells. We observedreplication of the TK vaccinia mutants in CIK cells (FIG. 1 a), however,the amount of infectious virus produced during the eclipse phase wasreduced relative to WR, and this served to accentuate the two-stepreplication kinetics.

The viral growth factor (VGF) gene product promotes cellular growthafter secretion from infected cells, by interacting with growth factorreceptors. VGF deletions have been shown to restrict viral replicationto cells with mutations in the Ras/MAPK/ERK pathway offering additionaltumor selectivity. Deletion of VGF was found to have a minimal effect onviral replication in CIK cells (FIG. 1 a), and infection of CIK cellswith a double deleted vaccinia virus (vvDD), containing deletions inboth TK and VGF produced almost no virus during the first 48 h (FIG. 1c). After this time, virus appeared to begin to accumulate withininfected CIK cells, before being released as the cells lyse. A highdegree of tumor selectivity has previously been demonstrated for thedouble deleted virus, both in vitro and in a variety of models in viva.For these reasons vvDD is used throughout the remainder of this study.

Further cell culture experiments were performed to examine therelationship between CIK cells and vvDD. A bioluminescence based cellsurvival assay was used to assess whether infected CIK cells can retainthe ability to recognize and destroy target ovarian tumor cell lines(FIG. 2 a). It was found that even after extended periods of infection(of at least 48 h), CIK cells remained functionally active and were ableto destroy tumor targets including human ovarian cancer cell lines. Inaddition, it was found that infection of a CIK-resistant ovarian tumorcell (SKOV-3) with vvDD sensitized them to subsequent CIK-mediatedkilling (FIG. 2 b, p=0.0076). CIK cells recognize NKG2D ligands ontarget cells, which are usually up-regulated under conditions ofcellular stress, such as are encountered within a tumor environment orfollowing viral infection. Two of the best characterized NKG2D ligandsin humans are MICA and MICB, and it was found that levels of theseproteins expressed on the surface of ovarian tumor cells corresponded totheir sensitivity to CIK cell-mediated killing (FIG. 2 c). MICA or MICBwas expressed on the surface of the sensitive UCI-101 cell line, but notthe resistant SKOV-3 cell line. However, infection of the SKOV-3 cellline with vvDD resulted in an increase in the percentage of cellsexpressing MICA or MICB, providing an explanation for the increasedsensitivity of this resistant tumor to CIK-mediated killing (FIG. 2 b).

In vivo non-invasive imaging experiments were then performed in order toassess both the ability of pre-infected CIK cells to traffic to ovariantumors and the subsequent biodistribution of virus compared to thatfollowing intravenous injection of virus alone. Conjugation of afluorescent dye (Cy5.5) to the CIK cells prior to intravenous deliveryenabled visualization of dye-labeled cells at the tumor site.Preinfection with vvDD did not affect the trafficking of CIK cells toUCI-101 tumors (Supplementary FIG. S1 a), this was verified with CIKcells labeled through expression of luciferase (FIG. 5 b). Since viralinfection was shown to increase NKG2D ligand expression and sensitizeSKOV-3 cells to CIK cytotoxicity in culture, we tested the effect ofpreinfected CIK cells on this otherwise resistant target cell.Cy5.5-labeled CIK cells, with or without pre-infection with vvDD, wereintravenously delivered to mice bearing SKOV-3 tumors. CIK cells alonedid not reduce the tumor burden, presumably due to lack of recognition,and no accumulation of CIK cells was observed at the tumor site (FIG. 5a). However, vvDD delivered along with the pre-infected CIK cells led toinfection of the tumors and subsequent MICA or MICB up-regulation andCIK accumulation (followed by release of further vvDD within the tumor).This was verified by immunofluorescence microscopy, with the sites ofCIK infiltration within the SKOV-3 tumor corresponding to areas of vvDDgene expression and MICA or MICB up-regulation (FIG. 6).

Using luciferase labeled vvDD and in vivo bioluminescence imaging, itwas possible to follow the biodistribution and duration of viral geneexpression for virus delivered alone or within CIK cells (FIG. 3 a).Initial infection patterns at 24 h post injection showed viral geneexpression within the lung, liver and spleen regardless of deliverymethod. However the ratios were different, with virus delivered aloneleading to signals predominantly from the spleen, and virus deliveredvia CIK cells leading to signal predominantly from the lungs. Thesystemic delivery potential of vaccinia and the vvDD virus has beendescribed previously. We observed tumor signal within 24 h followingintravenous delivery of virus alone, whereas the virus within CIK cellsdid not lead to detectable signal at the tumor site until 48 h postdelivery (which is similar to the time frame required for delivery ofCIK cells to tumors), by this time very little signal was detected inany organs other than the tumor. Equivalent levels of signal, indicativeof viral gene expression, were reached using both delivery methods by 5days post treatment (FIG. 3 b). Furthermore, signal from CIKcell-delivered virus was sustained within the tumor for longer periodsof time compared to virus alone (p=0.0079 at day 35). It was observedthat the bioluminescent signal within the tumor when virus was deliveredalone appeared more sporadic relative to the more uniform signalproduced from tumors treated with the combined therapy. This was alsoseen in an equivalent experiment using fluorescence imaging and a GFPexpressing strain of vvDD (FIG. 3 a).

A more uniform distribution of viral infection following CIK-mediateddelivery suggests penetration into the tumor, which along with sustainedviral gene expression, would lead to increased tumor destruction.Although vaccinia virus vectors alone can both reach the tumor andspread very efficiently from cell to cell, we observed that theirinitial infection was limited to cells surrounding the tumor vasculature(FIG. 3 c), and so they do not easily spread to more distant regions ofthe tumor. CIK cells however, once within the tumor environment arecapable of actively extravascating from the vasculature, and it waspredicted that preinfected CIK cells would carry oncolytic viruses intothe tumor mass. We observed that virus delivered via infected CIK cellsproduced a more uniform biodistribution of infection within the tumor,even at locations distant to the tumor vasculature (FIG. 3 c). Furtherimmunofluorescence microscopy was used to show that at 24 h postdelivery infected CIK cells could be found within the tumor (FIG. 7),demonstrating that they can deliver the viral agent to the tumor. By 72h post treatment, initial sites of tumor cell infection can be seen atthe edge of the tumor and surrounding the vasculature, however, infectedCIK cells can be seen at locations distant to these sites of preliminaryinfection (FIG. 3 d, arrows).

Lastly to demonstrate synergistic efficacy the survival of animalsbearing intraperitoneal ovarian tumors was followed after treatment viathe tail vein with a single dose of either agent alone or with thecombination biotherapy (FIG. 4 a). CIK cells alone were able tomarginally extend survival in the mice with sensitive UCI-101 tumors buthad no effect in mice with the resistant SKOV-3 tumor. vvDD was alsoable to marginally increase survival in both these models, with someanimals showing complete responses (FIG. 4 a). However, the combinationtherapy was capable of producing dramatically increased survival(p=<0.01) compared to either therapy alone, with 100% complete responsesamong the animals bearing UCI-101 tumors. The combination therapy alsodisplayed increased efficacy against SKOV-3 tumors compared to just thevvDD vector (p=0.0379), despite the fact that the CIK cells alone had noeffect against these tumors. Plots of the tumor burden in individualanimals (FIG. 4 b) indicated that single vvDD or CIK cell therapyagainst sensitive tumors produced an initial response but incompleteeradication of the tumor leading to subsequent relapse. Completeresponses were followed for up to 90 days without relapse.

Here we combine two biological therapies in such a way that we are ableto harness the benefits of both and produce a new combination therapythat far exceeds the effectiveness of either alone. CIK cells were usedto systemically deliver the oncolytic vaccinia virus, vvDD, efficientlyand specifically to the site of the tumor. The oncolytic virus can thenact to increase the tumor cell killing potential of the CIK cells. Inaddition to this however, we have found that the virus does not affectCIK cell function, but can sensitize tumor cells to CIK cell-mediatedkilling. Finally the CIK cells can transport the virus deep within thetumor, producing a more uniform biodistribution of viral infection oftumor cells within the cancer. The resulting targeted, biologicaltherapy therefore displays systemic delivery potential, minimaltoxicities and has significant anti-tumor effects.

Methods.

Cells and Viruses. Human ovarian cancer cell lines were SKOV3 (obtainedfrom ATCC) and UCI-101. Stably transduced versions of both cell lineswere produced by retroviral infection in order to produce cellsexpressing click beetle red luciferase and puromycin (for selection).Growth characteristics of the transfected cells were compared to theparental strains in vitro to verify no effects due to the retroviralinsertion. The human B cell lymphoma cell line (OCI-ly8) has beenpreviously characterized. The expansion of CIK cells has been describedpreviously by Lu & Negrin (1994) J Immunol 153, 1687-96. The strains ofvaccinia were wild type Western Reserve (obtained from ATCC), WesternReserve containing a single deletion in the viral thymidine kinase (TK)gene, and expressing luciferase containing a single deletion in the VGFgene and expressing beta-galactosidase and the double deleted virus(vvDD) containing deletions in both the thymidine kinase and VGF genesand expressing GFP from within the site of the TK gene or expressingluciferase from the TK gene (constructed by homologous recombination ofthe VGF deleted virus and the plasmid pSC-65 containing the fireflyluciferase gene from pGL3 (Promega)).

Viral Replication and Plaque Assays. Cell lines to be assayed wereinfected by addition of virus at the mulitiplicity of infection (MOI)(Plaque Forming Units (PFU)/cell) indicated. After 2 h of infection,media was changed and at various times either media alone was retainedor cells retained in PBS, or else media and cells were collectedtogether. Cells were lysed by three cycles of freeze/thaw in order torelease intracellular virus. Plaque assay was performed in 6-well plateson the BS-C-1 cell line (obtained from ATCC).

Cellular Cytotoxicity Assay. Tumor cell lysis by effector cells wasquantified by measuring the luciferase activity of surviving targetcells. Target cells expressing luciferase were plated into black-walled96-well plates at 1×10⁴ cells/well. Effector cells were then added atspecified effector to target ratios; all ratios (as well as target onlywells) were plated in triplicate and incubated for 4 h at 37° C., 5%CO₂. Luciferin was then added to each well (2 μl of 30 mg/ml luciferin(Xenogen Corp)) and light output (photons.second⁻¹/well) measured on anIVIS 50 imaging system (Xenogen Corp). Percent cytotoxicity was thendetermined relative to control wells (target only or target onlypre-treated with 70% ethanol).

FACS Assays. FACS assays were performed on samples of cells alone orcells preinfected with vvDD expressing GFP. Cells were then stained withanti-MICA/B antibody conjugated to PE (BD Pharmingen) at a dilution of1:200. Samples and controls were run on a FACScaliber (BectonDickinson).

Mouse tumor models. All mouse studies used CD1 nu/nu female mice aged 8to 10 weeks (obtained from Charles River). All animal studies wereperformed according to Stanford IACUC approval. Tumors were formed inthese animals by injection of 1×10⁶ tumor cells either subcutaneous orintraperitoneally. Animals injected subcutaneously were treated by asingle tail vein injection once palpable tumors of 50-100 mm³ wereformed (tumor size was followed by caliper measurement orbioluminescence imaging as indicated). Animals injectedintraperitoneally received luciferase labeled tumor cells only and wereimaged regularly by bioluminescence imaging and treated with a singleintravenous tail vein injection of therapeutic as described 3 days afterinjection of labeled tumor cells. Bioluminescence imaging was used toconfirm tumor cell light output was increasing prior to treatment.Non-invasive imaging assays. Both bioluminescence and fluorescenceimaging modalities were incorporated in order to image tumor cells,virus or CIK cell biodistribution. Animals were anesthetized andinjected with luciferin (150 mg/kg) 5 minutes prior to bioluminescenceimaging. Animals were placed on a warmed stage (37° C.) and imaged usingan IVIS 200 system (Xenogen Corp) for bioluminescence or Cy5.5 in vivofluorescence imaging and a Maestro (CRI) for GFP imaging. Wheneverfluorescent imaging was used, an initial background image was firsttaken for background subtraction immediately before the fluorescentimage, and no luciferin was injected. Appropriate filter sets were usedfor Cy5.5 and GFP imaging.

Labeling of CIK cells for imaging. CIK cells were either labeled withclick beetle red luciferase by retroviral transduction, or with Cy5.5NHS ester (Amersham Biosciences, GE).

Immunofluorescence Microscopy. Tumors from animal treated with Cy5.5labeled CIK cells and/or vvDD-GFP were frozen in OCT and sectioned (8-10microns). Tissue slices were fixed in acetone, blocked with 2% FCS andstained using primary antibodies to CD-31 or GFP (Molecular Probes) asindicated. Secondary staining was with AlexaFluor conjugated antibodies(Molecular Probes) and nuclear staining with Sytox Blue (MolecularProbes). Sections were then examined using a Zeiss Axiovert or a LeicaConfocal microscope.

Example 2 Cervical Cancer

The dual biotherapy as described above was effective in reducing tumorcell number for a variety of cervical cancer cells in vitro

A human cervical cancer cell line (SPEC) labeled with luciferase wastested in vivo. 5×10⁶ tumor cells were implanted intraperitoneally intoimmunodeficient (SCID) mice and allowed to grow for 7 days. Mice werethen treated with a single intraperitoneal injection of either PBS,1×10⁷ CIK cells or 1×10⁷ CIK cells pre-infected with a TK and VGF doubledeleted vaccinia virus. CIK cells alone transiently delayed the growthof these tumors. Vaccinia virus alone reduced tumor burden, which wasfollowed by relapse in 5 of 7 mice (i.e. 2 of the 7 mice displayed acomplete response). The dual biotherapy reduced tumor burden, where 5 ofthe 7 mice displayed a complete recovery from the tumor.

Example 3 Lung Cancer

A mouse non-small cell lung cancer cell line (CMT 64) was tested inimmunocompetent C57B/6 mice. Tumor cells were implanted subcutaneously,and allowed to grow until tumors reached 50-100 mm³. Animals were thentreated with a single tail vein injection of either PBS, 1×10⁷ PFU of TKdeleted vaccinia virus or 1×10⁷ CIK cells pre-infected with the samevaccinia virus. The dual biotherapy of the invention significantlyincreased the survival of these mice relative to virus alone (withmedian survival being 27 days in PBS treated mice; 38 days in virustreated mice and 59 days in dual biotherapy mice). 1 of 7 mice in dualbiotherapy group displayed a complete recovery from the tumor, and nonein the other treatment groups.

Example 4 Prostate Cancer

In a 3D tissue culture model (spheroid) of the PC3 human prostate cancercell line, it was seen that infected CIK cells deliver virus to thespheroids and subsequently kill the tumor cells. Spheroids contain 5-10000 tumor cells and were mixed with CIK cells infected with TK and VGFdeleted vaccinia virus, such that there were approximately 10 infectedCIK cells/spheroid. By 96 h after addition of the infected CIK cells,the spheroids were completely destroyed.

Example 5 Liver Cancer

The human liver cell lines (HepG2; Hep3B) were cultured in vitro, andshown to be efficiently killed by the dual biotherapy of the invention.Cell monolayers were mixed with CIK cells infected with a TK deletedvaccinia. Little viral infection of the cell layer was seen during theeclipse phase, followed by rapid viral infection of the cell layer(between 48 and 72 h post addition of infected CIK cells), and rapiddestruction of the cell layer.

Example 6 Lymphoma

A mouse lymphoma cell line (6780) was grown in immunocompetent animals(FVB) following intraperitoneal injection of 1×10⁷ tumor cells. It wasfound that subsequent intravenous administration of CIK cells (1×10⁷cells) had no effect; virus alone (1×10⁷ PFU of vaccinia with deletionsI the viral TK and VGF genes) briefly reduced tumor burden; and dualbiotherapy (1×10⁷ CIK cells pre-infected with the same virus) reducedtumor burden for an extended period of time.

The dual biotherapy of the invention also prevents relapse, due to bothefficient clearance of tumor cells and raising of an anti-tumor immuneresponse. Using a lymphoma cell line (6780) that expresses luciferase,and contains a tetracycline repressible myc oncogene (such that additionof doxycycline to the drinking water of the mice represses the oncogenemyc in the cell line, and the tumors regress).

The tumors were grown following intraperitoneal injection and were thenregressed by the addition of doxycycline until they were undetectable bybioluminescence imaging. The animals were then treated intravenouslywith a single injection of PBS; CIK (1×10⁷ cells); or dual biotherapy(1×10⁷ CIK cells pre-infected with TK and VGF deleted vaccinia virus),and doxycycline removed from their drinking water.

The control mice all relapsed within 7-10 days. The animals treated withCIK alone had a delay, relapsing from 10 to 90 days after cessation ofdoxycycline. In the dual biotherapy group, 4 of 7 mice relapsed late(10-90 days), the other 3 mice never relapsed. The data is shown in FIG.8.

The mice that did not relapse were subsequently challenged with the 6780lymphoma cells a second time (1×10⁷ 6780 cells injectedintraperitoneally 3 months after the treatment). The mice rejected thetumor cells, and showed an anti-tumor CTL response (splenocytes fromthese mice were stimulated with 6780 cells in culture and assayed forIFN-gamma production, significantly more splenocytes were activated inthese mice than in control mice).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

1. An isolated mammalian immune effector cell population, infected withan oncolytic virus.
 2. The cell population according to claim 1, whereinsaid cell population is human.
 3. The cell population according to claim1, wherein said immune effector cell is a T cell and/or natural killercell.
 4. The cell population according to claim 1, wherein said immuneeffector cells are expanded in in vitro culture.
 5. The cell populationaccording to claim 4, wherein said effector cell is a cytokine inducedkiller (CIK) cell.
 6. The cell population according to claim 3, whereinsaid oncolytic virus is a vaccinia virus.
 7. The cell populationaccording to claim 6, wherein said vaccinia virus comprises a mutationin the viral thymidine kinase gene.
 8. The cell population according toclaim 6, wherein said vaccinia virus comprises a mutation in the viralgrowth factor (VGF) gene.
 9. The cell population according to claim 1,wherein said replication of said oncolytic virus is in an eclipse phase.10. The cell population according to claim 1, further comprising apharmaceutically acceptable excipient.
 11. A method of treating cancerin a patient, the method comprising: administering to a cancer patientan effective amount of a mammalian immune effector cell populationinfected with an oncolytic virus.
 12. The method according to claim 11,wherein said cell population is human.
 13. The method according to claim11, wherein said immune effector cell is a T cell and/or natural killercell.
 14. The method according to claim 11, wherein said immune effectorcells are expanded in in vitro culture.
 15. The method according toclaim 14, wherein said effector cell is a cytokine induced killer (CIK)cell.
 16. The method according to claim 14, wherein said oncolytic virusis a vaccinia virus.
 17. The method according to claim 16, wherein saidvaccinia virus comprises a mutation in the viral thymidine kinase gene.18. The method according to claim 16, wherein said vaccinia viruscomprises a mutation in the viral growth factor (VGF) gene.
 19. Themethod according to claim 11, wherein said administering is performedwhen replication of said oncolytic virus is in an eclipse phase.